Whole blood or blood plasma is collected from human beings for a variety of reasons. These can include analytical reasons such as analysis of the blood for foreign matter, or can be for reasons such as the collection and distribution to hospitals for use during, for example, surgical operations.
One of the problems associated with collecting blood, is that when blood leaves the body, it has a tendency to coagulate. Because of this, it is common to collect the blood into a receptacle that contains an anticoagulant. This anticoagulant will typically prevent or inhibit the reaction that clots (coagulates) blood. Examples of anticoagulants include trypsin inhibitor, hirudin, and heparin, heparin being the most common anticoagulant for blood. As used herein, the term blood refers to blood in its various forms, including whole blood or blood plasma.
Heparin is a mucopolysaccharide composed of sulfated D-glucosamine and D-glucuronic acid. Heparin is a heterogenous compound and it is estimated that the molecular weight can vary from about 6,000 to about 20,000. The primary anticoagulation function of heparin is believed to be the prevention of the formation of thrombin in the blood clotting process.
Heparin is commercially available in a wide range of activity concentrations, usually expressed in U.S.P. units per milligram, or units/mg. For example, the activity concentration of heparin can range from about 140 units/mg to about 250 units/mg.
Traditionally, a concentration of from about 50 to about 400 U.S.P. units of heparin activity per milliliter of blood (units/ml) is used to anticoagulate a blood sample. As used herein, the term "unit" is used to refer to the United States Pharmacopoeia (U.S.P.) unit of heparin activity. Another common measurement of activity is the international unit, or IU. The international unit is believed to be about 6.4 percent larger then the U.S.P. unit.
The present inventors believe that one of the many reasons that high concentrations (i.e. 50 units/ml of blood and higher) of heparin have been utilized in blood sampling receptacles, is that there is a surface effect, particularly with glass, that promotes the coagulation of blood. Therefore, it was desirable to use high concentrations of heparin in glass blood sampling receptacles to inhibit the coagulation of the blood. Further, the use of heparin did not substantially bias any subsequent analyses, so there was little motivation to seek a reduction in the concentration.
Today, blood is often drawn from a patient into a plastic receptacle for blood gas analysis. Additionally, advances in blood analyzing technology have enabled medical personnel to measure the free ion concentration in the blood, including free calcium ions. For example, devices for measuring free calcium in blood are available from Nova Biomedical Corporation, Waltham, Mass., ABX Corporation, Horsham, Pa., and Ciba-Corning, Severna Park, Md.
The need to accurately measure free calcium ion in a patient's blood is important in a number of instances. For example, medical personnel require accurate free calcium ion measurements for patients with hypertension to predict which patients will benefit from an increase in the oral intake of calcium. Free calcium ion is also routinely measured when a major medical decision may be influenced by the patient's calcium status, for example, decisions relating to endocrine disorders. Further, sick newborn children are susceptible to losing calcium easily and may not readily reabsorb it, so accurate measurements are required.
However, heparin compounds are known to chelate free calcium ions, and therefore bias the measurement of free calcium ions. As a result, when traditional concentrations of heparin are used for anticoagulation in a collected blood sample, the free calcium ion measurement can be significantly biased.
Numerous techniques have been suggested to overcome this particular problem. U.S. Pat. No. 4,687,000 by Eisenhardt et al., issued Aug. 18, 1987, discloses a method for treating blood with an anticoagulant, preferably heparin, and compensating for the anticoagulant binding of several cation species by adding compensating amounts of the cations to the blood sample. The concentration of the cation species thus remains constant in the blood sample and may be determined by subsequent analysis. However this procedure necessitates the determination of the extent that cation species are bound, requiring experimentation and theoretical assumption.
Another approach to solving the chelation problem is to lower the concentration of heparin in the blood sample so that the chelating effects on the free ions are minimized.
For example, in "Facilitated Determination of Ionized Calcium," by Urban et al., Clinical Chemistry, Vol. 31, No. 2, pp. 264-266 (1985), such a method for determining the amount of ionized calcium in blood is disclosed. In the experiment, sodium heparin was utilized in a concentration of between about 10 and about 25 IU/ml of whole blood and a calcium heparin preparation in a concentration of about 20 IU/ml of whole blood was also utilized It is disclosed that the use of sodium heparin in a concentration less than 5 IU/ml yielded free ionized calcium measurements similar to those obtained for a reference serum. However, there was frequent clogging of the electrode system, indicating poor anticoagulation action. Further, it is disclosed that the proper heparin dilution was difficult to prepare.
In "Heparinization of Samples for Plasma Ionized Calcium Measurement," by Heining et al., in Critical Care Medicine, Vol. 16, No. 1, pp. 67-68 (1988), it is disclosed that when heparin is used in concentrations greater than about 10 IU/ml of blood, complexes form with the calcium ions. It is concluded that for measurement of plasma-free ionized calcium, blood samples should be heparinized in a quantified fashion to insure that the heparin concentration does not exceed 10 IU/ml of whole blood. If the concentration exceeds this level, it is alleged that falsely low readings will be obtained. However, such low concentrations of heparin are difficult to control, particularly outside of the research laboratory. For instance, a three cubic centimeter (3 cm.sup.3) syringe may have from 0.5 to 3.0 cubic centimeters of blood drawn into it when used in practical situations. Deviations in the amount of heparin activity in the blood sampling device may result in a significant increase in the concentration of heparin activity in the blood sample and lead to a biasing of free ion measurements.
Hence, due to the biasing effect on the measurement of ions, it is preferable to use a very low unit dosage of heparin as an anticoagulant to minimize the biasing effect. The heparin may be introduced into the blood in many forms, including as a solid pledget or as a liquid. Solid pledgets are preferred since excess liquid heparin can result in over-dilution of the blood sample and accentuated binding of ions, such as calcium, to the heparin. However, the production of solid pledgets containing low unit dosages has been found to be extremely difficult to implement.
A pledget is a single unit dosage of heparin in tablet form. A process for the manufacture of a pledget is described, for example, in commonly-owned U.S. Pat. No. 4,521,975 by Bailey, issued June 11, 1985. This patent describes a process for the production of a pledget wherein the predetermined unit dosage is formed by a lyophilizing process.
However, the pledgets produced by the process disclosed by Bailey are used to anticoagulate blood samples such that the heparin concentration is from about 100 to about 200 units/ml of blood. Recent advances in blood-analyzing technology have dictated that much lower levels of heparin be used.
If the pledget is made purely of heparin, and the heparin has an average activity of about 180 units/mg of heparin, a pledget providing 2.8 U.S.P. units of heparin would have a mass of about 16 micrograms, a volume of approximately 11 microliters, yielding a density of only about 1.5 mg/ml. Such a small size with such low density is very difficult to manufacture, particularly on a commercial scale. It would be advantageous to manufacture larger pledgets, which are more easily handled by existing machinery.
One possible solution is to place the heparin compound on a carrier body. U.S. Pat. No. 4,687,000 by Eisenhardt, et al., discussed hereinabove, describes carrier bodies used with heparin compositions. It is disclosed that the carrier body may, for example, be made of material such as filter paper, synthetic fibers, glass fibers, mineral fibers, or the like. For example, filter paper is used to absorb the heparin solution and create a carrier body with the desired level of heparin. However, one of the problems with using these materials is that it is very difficult to control the concentration of heparin in any individual dose. Further, the disclosed materials do not substantially dissolve and may interfere with the proper operation of the blood sampling or analyzing device.
U.S. Pat. No. 4,479,799 by Thiel, issued on Oct. 30, 1984, discloses that one method for introducing an anticoagulant such as heparin into a blood sample is to place an anticoagulant tablet in the hub of the needle of the syringe used to obtain the blood sample from the patient. It is disclosed that the tablets may comprise a heparin salt, a tablet binder and a pH controlling substance. It is disclosed that the use of these tablets requires a mixing step after the blood is drawn into the syringe and that the tablet binder and pH controlling substance require added cost and additional manufacturing complexities. Thiel then discloses a new process for producing a web of heparin that may be placed directly into the hub of a needle through which blood to be analyzed is drawn. Thus, although Thiel recognizes problems associated with the manufacturing of a tablet, Thiel does not address a solution whereby existing manufacturing processes and apparatus can be used, nor does Thiel address the special problem associated with producing pledgets with low unit dosages of heparin.
U.S. Pat. No. 4,371,516 by Gregory et al., issued Feb. 1, 1983, discloses articles for carrying chemicals, particulary pharmaceutical dosages, which dissolve rapidly in water. The articles include a carrier material such as hydrolysed gelatin, dextran, dextrin, or alginates. Gregory et al., also disclose a process for preparing the articles by subliming solvent from a composition comprising the pharmaceutical in a solution of the carrier material in a solvent. The carrier bodies disclosed by Gregory et al., are rather large, about 0.75 ml in volume, and comprise a high concentration of pharmaceutical. It would be beneficial to produce a carrier body more amenable to use in a small syringe, such as a one cubic centimeter syringe. However, smaller bodies are much more likely to stick in the container in which they are produced. Gregory et al. attempts to address this problem in the larger tablets by using a surfactant.
Gregory et al. also disclose that the solution to be sublimed has a solids concentration of about 73 mg/ml or higher. Solutions with these high solids concentrations give rise to tablets that are very difficult to redissolve in solvents such as blood, particularly when rapid mixing is desired.
Pledgets for anticoagulating blood samples should preferably contain a relatively low concentration of heparin to be useful when it will be desirable to measure free ions in the blood, particularly calcium ions. However, the manufacture of extremely small pledgets of heparin is difficult to implement. The small size makes it difficult to remove the pledget from its mold without inflicting damage to the pledget. And at very small sizes, forces such as static electricity become significant and further complicate handling.
It is therefore desirable to increase the size of the pledget without significantly increasing the heparin activity content. It has been suggested that filtering paper or mineral fibers may be used as a carrier body, however, these may interfere with the proper operation of the blood sampling device, since these materials do not dissolve and it is difficult to accurately control the unit dosage.
The use of materials such as hydrolysed gelatin or polysaccharrides such as dextran, dextrin and alginates as pharmaceutical carrier bodies for large tablets (i.e. 0.5 ml and larger) has also been described. However, these tablets are too large to use in standard blood sampling devices, which require pledget sizes less than about 0.075 ml.
The production of smaller pledgets, i.e. less than about 0.075 ml, presents special problems. The above-mentioned carrier body materials tend to stick to mold walls during formation of the pledget, and hence, small pledgets are often damaged by attempts to remove them from the mold. Irregularities, such as nicks, burrs, or foreign matter on the mold wall compound this problem by making the smaller pledget more difficult to remove. It would also be highly desirable to eliminate the problems of the pledget sticking in the mold without resorting to additional chemicals, such as surfactants.
The production of such a pledget, for example by lyophilization, also requires that particular problems be addressed. The solution from which the pledget is derived must contain a sufficient amount of filler to retain adequate strength in the pledget, while being low enough in density to permit rapid dissolution in the blood sample. It would also be highly desirable to eliminate the problems of the pledget sticking in the mold without resorting to additional chemicals, such as surfactants.